Adenosine receptors (ARs) are classified into different subtypes (A1, A2A, A2B and A3Rs) and are expressed in neurons and skeletal muscles. In central nervous system adenosine acts as neuromodulator but its effect in skeletal muscle is still unclear. Here we investigated the possible interplay between ARs and embryonic nicotinic acetylcholine receptor (nAChR) expressed in denervated as well as in developing skeletal muscles. Electrophysiological experiments were performed in oocytes injected with denervated skeletal muscle membranes and in differentiating mouse myotubes grown in vitro. The nACh-currents were recorded before and after adding the AR agonist NECA (100 μM) or the antagonist CGS15943 (100 nM). In injected oocytes, we observed that the activation of ARs reduced the nACh-current rundown and slowed the nACh-current decay, while the AR inhibition elicited the opposite effect. In developing myotubes, recordings of single nAChR channel activity in cell-attached configuration revealed that NECA significantly increased the mean open time while CGS15943 reduced it; both agents did not affect the ion channel conductance. In parallel, Ca2+ imaging experiments showed that NECA increased and CGS15943 blocked the [Ca2+]i spiking activity sustained by the autocrine activation of the nAChRs. Our preliminary data indicate a modulatory effect mediated by activation of ARs on the embryonic nAChR channels, expressed by denervated and developing skeletal muscles. We hypothesize a potential novel role for ARs in the regulation of AChR-mediated events during skeletal muscle differentiation/regeneration.